This invention relates to xe2x80x9ctargetablexe2x80x9d vector particles. More particularly, this invention relates to vector particles which include a receptor binding region that binds to a receptor of a target cell of a human or non-human animal.
Vector particles are useful agents for introducing gene(s) or DNA (RNA) into a cell, such as a eukaryotic cell. The gene(s) is controlled by an appropriate promoter. Examples of vectors which may be employed to generate vector particles include prokaryotic vectors, such as bacterial vectors; eukaryotic vectors, including fungal vectors such as yeast vectors; and viral vectors such as DNA virus vectors, RNA virus vectors, and retroviral vectors. Retroviral vectors which have been employed for generating vector particles for introducing genes or DNA (RNA) into a cell include Moloney Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus and Harvey Sarcoma Virus. The term xe2x80x9cintroducingxe2x80x9d as used herein encompasses a variety of methods of transferring genes or DNA (RNA) into a cell, such methods including transformation, transduction, transfection, and infection.
Vector particles have been used for introducing DNA (RNA) into cells for gene therapy purposes. In general, such a procedure involves obtaining cells from a patient and using a vector particle to introduce desired DNA (RNA) into the cells and then providing the patient with the engineered cells for a therapeutic purpose. It would be desirable to provide alternative procedures for gene therapy. Such an alternative procedure would involve genetically engineering cells in vivo. In such a procedure, a vector particle which includes the desired DNA (RNA) would be administered directly to the target cells of a patient in vivo.
It is therefore an object of the present invention to provide gene therapy by introduction of a vector particle, such as, for example, a retroviral vector particle, directly into a desired target cell of a patient.
In accordance with an aspect of the present invention, there is provided a retroviral vector particle which includes a receptor binding region or ligand that binds to a receptor of a target cell. The receptor of the target cell is a receptor other than the amphotropic cell receptor.
Retroviruses have an envelope protein which contains a receptor binding region. Applicants have found that retroviruses can be made xe2x80x9ctargetablexe2x80x9d to a specific type of cell if the receptor binding region of the retrovirus, which may be amphotropic, ecotropic, or xenotropic, among other types, is modified such that the receptor binding region of the envelope protein includes a receptor binding region which binds to a receptor of a target cell. For example, at least a portion of the receptor binding region of the envelope protein of the retrovirus is deleted and replaced with a receptor binding region or a ligand which binds to a receptor of a target cell. Thus, there is provided a retroviral vector wherein at least a portion of the DNA (RNA) which encodes the receptor binding region of the envelope protein of the retrovirus has been deleted and replaced with DNA (RNA) encoding a receptor binding region or a ligand which binds to a receptor of a target cell.
In one embodiment, the retrovirus is a murine leukemia virus.
The envelope of murine leukemia viruses includes a protein known as gp70. Such viruses can be made xe2x80x9ctargetablexe2x80x9d to a specific type of cell if a portion of the gp70 protein is deleted and replaced with a receptor binding region or a ligand which binds to a receptor of a target cell. Thus, in a preferred embodiment, there is provided a retroviral vector wherein a portion, but not all, of the DNA (RNA) encoding gp70 protein has been deleted and replaced with DNA (RNA) encoding a receptor binding region or a ligand which binds to a receptor of a target cell.
In general, gp70 protein includes the following regions: (i) the secretory signal or xe2x80x9cleaderxe2x80x9d sequence; (ii) the receptor binding domain; (iii) the hinge region; and (iv) the body portion. Preferably, at least a portion of the DNA (RNA) encoding the receptor binding domain of gp70 protein is deleted and replaced with DNA (RNA) encoding a receptor binding region or a ligand which binds to a receptor of a target cell. More preferably, DNA (RNA) encoding the entire receptor binding domain of gp70 protein is deleted and replaced with DNA (RNA) encoding a receptor binding region or a ligand which binds to a receptor of a target cell. In another embodiment, DNA. (RNA) encoding the entire receptor binding domain of gp70 protein, plus all or a portion of the DNA (RNA) encoding the hinge region of gp70 protein is deleted and replaced with DNA (RNA) encoding a receptor binding region or a ligand of a target cell.
The gp70 protein may be derived from an ecotropic murine leukemia virus, a xenotropic murine leukemia virus, or an amphotropic murine leukemia virus. Ecotropic gp70 (or eco gp70) (SEQ ID NO:1) is a protein having 469 amino acids, and is encoded by (SEQ ID:2). Amino acid residues 1-33 constitute the leader sequence; amino acid residues 34-263 constitute the receptor binding domain; amino acid residues 264-312 constitute the hinge region; and amino acid residues 313-469 constitute the body portion. Preferably, DNA (RNA) encoding at least a portion of the receptor binding region is removed and replaced with DNA (RNA) encoding a receptor binding region or a ligand which binds to a receptor of a target cell. More preferably, DNA (RNA) encoding some or all of amino acid residues 34 to 263 (i.e., the receptor binding domain) is removed and replaced with DNA (RNA) encoding a receptor binding region or a ligand which binds to a receptor of a target cell.
Xenotropic gp70 (or xeno gp70) (SEQ ID NO:3) has 443 amino acid residues and is encoded by (SEQ ID NO:4). Amino acid residues 1-30 constitute the leader sequence; amino acid residues 31-232 constitute the receptor binding domain; amino acid residues 233-286 constitute the hinge region; and amino acid residues 287-443 constitute the body portion. Preferably, DNA (RNA) encoding at least a portion of the receptor binding region is removed and replaced with DNA (RNA) encoding a receptor binding region or a ligand which binds to a receptor of a target cell. More preferably, DNA (RNA) encoding some or all of amino acid residues 31 to 232 is removed and replaced with DNA (RNA) encoding a receptor binding region or a ligand which binds to a receptor of a target cell.
Target cells to which the retroviral vector particle may bind include, but are not limited to, liver cells, T-cells, lymphocytes, endothelial cells, T4 helper cells, and macrophages. In one embodiment, the retroviral vector particle binds to a liver cell, and in particular to hepatocytes. To enable such binding, the retroviral vector particle contains a chimeric protein encoded by DNA (RNA) in which at least a portion of the DNA (RNA) encoding the receptor binding domain of gp70 protein is removed and is replaced with DNA (RNA) which encodes a protein which binds to an asialoglycoprotein receptor (or ASG-R) of hepatocytes.
Proteins which bind to the asialoglycoprotein receptor of liver cells include, but are not limited to, asialoglycoproteins such as, for example, alpha-1-acid glycoprotein (AGP), also known as orosomucoid, and asialofetuin. AGP is a natural high-affinity ligand for ASG-R. The asialoglycoprotein receptor, or ASG-R, is expressed only by hepatocytes. The receptor is present at about 3xc3x97105 copies per cell, and such receptors have a high affinity for asialoglycoproteins such as AGP. Thus, the engineering of retroviral vector particles to contain asialoglycoprotein in place of the natural receptor binding domain of gp70 generates retroviral vector particles which bind to the asialoglycoprotein receptor of hepatocytes, which provides for an efficient means of transferring genes of interest to liver cells.
Cell lines which generate retroviral vector particles that are capable of targeting the hepatocyte""s asialoglycoprotein receptor without the removal of the particle""s terminal sialic acid groups by neuraminidase treatment, can be developed by selection with the cytotoxic lectin, wheat germ agglutinin (WGA). Cell lines which express the retroviral proteins gag and pol become retroviral vector packaging cell lines after they are transfected with the plasmids encoding chimeric envelope genes. These cell lines express the corresponding chimeric gp 70 glycoproteins. Upon exposure to successively higher concentrations of WGA, the outgrowth of cells which synthesize glycoproteins that lack terminal sialic acid groups, is favored. (Stanley, et al., Somatic Cell Genetics, Vol. 3, pgs. 391-405 (1977)). This selection permits the isolation of cells which synthesize oligosaccharides terminating in galactosyl sugar groups. Such cells will allow the construction of packaging cell lines that are capable of generating retroviral vector particles which target the asialoglycoprotein receptor. It is also possible to select subpopulations of packaging cells which have other distinct glycotypes, such cells yielding viral vectors that potentially are capable of targeting cells other than hepatocytes. Macrophages, for example, express unique, high-mannose receptors. The PHA-resistant subpopulation will have N-linked oligosaccharides which terminate in high-mannose groups (Stanley, et al., In Vitro, Vol. 12, pgs. 208-215 (1976)). Therefore, such a cell population will be capable of producing viral vector particles capable of targeting macrophoges via this receptor. Cells with mutant glycotypes which synthesize other novel oligosaccharides after selection with other cytotoxic lectins may also prove to be useful in targeting vector particles to other cell types such as lymphocytes or endothelial cells.
In another embodiment, the receptor binding region is a receptor binding region of a human virus. In one embodiment, the receptor binding region of a human virus is a hepatitis B virus surface protein binding region, and the target cell is a liver cell.
In another embodiment, the receptor binding region of a human virus is the gp46 protein of HTLV-I virus, and the target cell is a T-cell.
In yet another embodiment, the receptor binding region of a human virus is the HIV gp120 CD4 binding region, and the target cell is a T4 helper cell.
In one embodiment, the retroviral vector may be of the LN series of vectors, as described in Bender, et al., J. Virol., Vol. 61, pgs. 1639-1649 (1987), and Miller, et al., Biotechniques, Vol. 7, pgs. 98-990 (1989).
In another embodiment, the retroviral vector includes a multiple restriction enzyme site, or multiple cloning site. The multiple cloning site includes at least four cloning, or restriction enzyme sites, wherein at least two of the sites have an average frequency of appearance in eukaryotic genes of less than once in 10,000 base pairs; i.e., the restriction product has an average size of at least 10,000 base pairs.
In general, such restriction sites, also sometimes hereinafter referred to as xe2x80x9crarexe2x80x9d sites, which have an average frequency of appearance in eukaryotic genes of less than once in 10,000 base pairs, contain a CG doublet within their recognition sequence, such doublet appearing particularly infrequently in the mammalian genome. Another measure of rarity or scarcity of a restriction enzyme site in mammals is its representation in mammalian viruses, such as SV40. In general, an enzyme whose recognition sequence is absent in SV40 may be a candidate for being a xe2x80x9crarexe2x80x9d mammalian cutter.
Examples of restriction enzyme sites having an average frequency of appearance in eukaryotic genes of less than once in 10,000 base pairs include, but are not limited to the NotI, SnaBI, SalI, XhoI, ClaI, SacI, EagI, and SmaI sites. Preferred cloning sites are selected from the group consisting of NotI, SnaBI, SalI, and XhoI.
Preferably, the multiple cloning site has a length no greater than about 70 base pairs, and preferably no greater than about 60 base pairs. In general, the multiple restriction enzyme site, or multiple cloning site is located between the 5xe2x80x2 LTR and 3xe2x80x2 LTR of the retroviral vector. The 5xe2x80x2 end of the multiple cloning site is no greater than about 895 base pairs from the 3xe2x80x2 end of the 5xe2x80x2 LTR, preferably at least about 375 base pairs from the 3xe2x80x2 end of the 5xe2x80x2 LTR. The 3xe2x80x2 end of the multiple cloning site is no greater than about 40 base pairs from the 5xe2x80x2 end of the 3xe2x80x2 LTR, and preferably at least 11 base pairs from the 5xe2x80x2 end of the 3xe2x80x2 LTR.
Such vectors may be engineered from existing retroviral vectors through genetic engineering techniques known in the art such that the retroviral vector includes at least four cloning sites wherein at least two of the cloning sites are selected from the group consisting of the NotI, SnaBI, SalI, and XhoI cloning sites. In a preferred embodiment, the retroviral vector includes each of the NotI, SnaBI, SalI, and XhoI cloning sites.
Such a retroviral vector may serve as part of a cloning system for the transfer of genes to such retroviral vector. Thus, there may be provided a cloning system for the manipulation of genes in a retroviral vector which includes a retroviral vector including a multiple cloning site of the type hereinabove described, and a shuttle cloning vector which includes at least two cloning sites which are compatible with at least two cloning sites selected from the group consisting of NotI, SnaBI, SalI, and XhoI located on the retroviral vector. The shuttle cloning vector also includes at least one desired gene which is capable of being transferred from said shuttle cloning vector to said retroviral vector.
The shuttle cloning vector may be constructed from a basic xe2x80x9cbackbonexe2x80x9d vector or fragment to which are ligated one or more linkers which include cloning or restriction enzyme recognition sites. Included in the cloning sites are the compatible, or complementary cloning sites hereinabove described. Genes and/or promoters having ends corresponding to the restriction sites of the shuttle vector may be ligated into the shuttle vector through techniques known in the art.
The shuttle cloning vector may be employed to amplify DNA sequences in prokaryotic systems. The shuttle cloning vector may be prepared from plasmids generally used in prokaryotic systems and in particular in bacteria. Thus, for example, the shuttle cloning vector may be derived from plasmids such as pBR322; pUC18; etc.
Such retroviral vectors are transfected or transduced into a packaging cell line, whereby there are generated infectious vector particles which include the retroviral vector. In general, the vector is transfected into the packaging cell line along with a packaging defective helper virus which includes genes encoding the gag and pol, and the env proteins of the virus. Representative examples of packaging cell lines include, but are not limited to, the PE501 and PA317 cell lines disclosed in Miller, et al., Biotechniques, Vol. 7 pgs. 980-990 (1989).
The vector particles generated from the packaging cell line, which are also engineered with a protein containing a receptor binding region that binds to a receptor of a target cell, said receptor being other than the amphotropic cell receptor, are targetable, whereby the receptor binding region enables the vector particles to bind to a target cell. The retroviral vector particles thus may be directly administered to a desired target cell ex vivo, and such cells may then be administered to a patient as part of a gene therapy procedure.
Although the vector particles may be. administered directly to a target cell, the vector particles may be engineered such that the vector particles are xe2x80x9cinjectablexe2x80x9d as well as targetable; i.e., the vector particles are resistant to inactivation by human serum, and thus the targetable vector particles may be administered to a patient by intravenous injection, and travel directly to a desired target cell or tissue without being inactivated by human serum.
The envelope of retroviruses also includes a protein known as p15E, and Applicants have found that retroviruses are susceptible to inactivation by human serum a a result of the action of complement protein(s) present in serum on the p15E protein portion of the retrovirus. Applicants have further found that such retroviruses can be made resistant to inactivation by human serum by mutating such p15E protein.
In one embodiment, therefore, the retroviral vector is engineered such that a portion of the DNA (RNA) encoding p15E protein (shown in the accompanying sequence listing as SEQ ID NO:7), has been mutated to render the vector particle resistant to inactivation by human serum; i.e., at least one amino acid but not all of the amino acids of the p15E protein has been changed, or mutated.
p15E protein is a viral protein having 196 amino acid residues. In viruses, sometimes all 196 amino acid residues are present, and in other viruses, amino acid residues 181 to 196 (known as the xe2x80x9crxe2x80x9d peptide), are not present, and the resulting protein is the xe2x80x9cmaturexe2x80x9d form of p15E known as p12E. Thus, viruses can contain both the p15E and p12E proteins. p15E protein is anchored in the viral membrane such that amino acid residues residues 1 to 134 are present on the outside of the virus. Although this embodiment of the present invention is not to be limited to any of the following reasoning, Applicants believe complement proteins may bind to this region whereby such binding leads to inactivation and/or lysis of the retrovirus. In particular, the p15E protein includes two regions, amino acid residues 39 to 61 (sometimes hereinafter referred to as region 1), and amino acid residues 101 to 123 (sometimes hereinafter referred to as region 2), which Applicants believe have an external location in the three-dimensional structure of the p15E protein; i.e., such regions are directly exposed to human serum. Region 2 is a highly conserved region in many retroviruses, even though the amino acid sequences of this region are not identical in all retroviruses. Such regions are complement binding regions. Examples of complement proteins which may bind to the complement binding regions are ClS and ClQ, which bind to regions 1 and 2.
In order to inactivate the retrovirus, complement proteins bind to both region 1 and region 2. Thus, in a preferred embodiment, at least one portion of DNA encoding a complement binding region of p15E protein has been mutated. Such a mutation results in a change of at least one amino acid residue of a complement binding region of p15E protein. The change in at least one amino acid residue of a complement binding region of p15E protein prevents binding of a complement protein to the complement binding region, thereby preventing complement inactivation of the retrovirus. In one embodiment, at least one amino acid residue in both complement binding regions of p15E protein is changed, whereas in another embodiment, at least one amino acid residue in one of the complement binding regions is changed.
It is to be understood, however, that the entire DNA sequence encoding p15E protein cannot be mutated because such a change renders the vectors unsuitable for in vivo use.
In one embodiment, the mutation of DNA (RNA) encoding p15E protein may be effected by deleting a portion of the p15E gene, and replacing the deleted portion of the p15E gene, with fragment(s) or portion(s) of a gene encoding another viral protein. In one embodiment, one portion of DNA encoding the p15E protein is replaced with a fragment of the gene encoding the p21 protein, which is an HTLV-I transmembrane protein. HTLV-I virus has been found to be resistant to binding by complement proteins and thus HTLV-I is resistant to inactivation by human serum (Hoshino, et al., Nature, Vol. 310, pgs. 324-325 (1984)). Thus, in one embodiment, there is also provided a retroviral vector particle wherein a portion of the p15E protein has been deleted and replaced with a portion of another viral protein, such as a portion of the p21 protein.
p21 protein (as shown in the accompanying sequence listing as SEQ ID NO:8) is a protein having 176 amino acid residues, and which, in relation to p15E, has significant amino acid sequence homology. In one embodiment, at least amino acid residues 39 to 61, and 101 to 123 are deleted from p15E protein, and replaced with amino acid residues 34 to 56 and 96 to 118 of p21 protein. In one alternative, at least amino acid residues 39 to 123 of p15E protein are deleted and replaced with amino acid residues 34 to 118 of p21 protein.
In another embodiment, amino acid residues 39 to 69 of p15E protein are deleted and replaced with amino acid residues 34 to 64 of p21 protein, and amino acid residues 96 to 123 of p15E protein are deleted and replaced with amino acid residues 91 to 118 of p21 protein.
Vector particles generated from such packaging lines, therefore, are xe2x80x9ctargetablexe2x80x9d and xe2x80x9cinjectable,xe2x80x9d whereby such vector particles, upon administration to a patient, travel directly to a desired target cell or tissue.
The targetable vector particles are useful for the introduction of desired heterologous genes into target cells ex vivo. Such cells may then be administered to a patient as a gene therapy procedure, whereas vector particles which are targetable and injectable may be administered in vivo to the patient, whereby the vector particles travel directly to a desired target cell.
Thus, preferably, the vectors or vector particles of the present invention further include at least one heterologous gene. Heterologous or foreign genes which may be placed into the vector or vector particles include, but are not limited to, genes which encode cytokines or cellular growth factors, such as lymphokines, which are growth factors for lymphocytes. Other examples of foreign genes include, but are not limited to, genes encoding Factor VIII, Factor IX, tumor necrosis factors (TNF""s), ADA, ApoE, ApoC, and Protein C.
The vectors of the present invention include one or more promoters. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniques, Vol. 7, No. 9, pgs 980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and B-actin promoters). Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, TK promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.
The vectors of the present invention may contain regulatory elements, where necessary to ensure tissue specific expression of the desired heterologous gene(s), and/or to regulate expression of the heterologous gene(s) in response to cellular or metabolic signals.
Although the invention has been described with respect to retroviral vector particles, other viral vector particles (such as, for example, adenovirus, adeno-associated virus, and Herpes Simplex virus particles), or synthetic particles may be constructed such that the vector particles include a receptor binding region that binds to a receptor of a target cell, wherein the receptor of a human target cell is other than the amphotropic cell receptor. Such vector particles are suitable for in vivo administration to a desired target cell.
Advantages of the present invention include the ability to provide vector particles which may be administered directly to a desired target cell or tissues, whereby desired genes are delivered to the target cell or tissue, whereby the target cell or tissue may produce the proteins expressed by such genes.
This invention will now be described with respect to the following examples; however, the scope of the present invention is not intended to be limited thereby.